A comprehensive program in Ca site design is proposed to directly address the primary objective of this program project: bridging the gap between description of structures and comprehension of activity of metalloproteins. The broad, long-term objective of our research is to develop the ability to create new functional Ca sites in proteins for biomedical research and medical applications. One primary objective of our project is to understand the variation in the responses to Ca binding of EF-hand Ca-binding proteins (CaBPs). These proteins have been selected because they have central roles in nearly all Ca signaling pathways and consequently, are associated with a wide-range of effects on health and disease (cell cycle, cancer, etc.). In Aim 1, in-depth comparative analyses of EF-range of effects on health and disease (cell cycle, cancer, etc.). In Aim 1, in-depth comparative analysis of EF-hand CaBPs will be used to develop hypotheses about what structural factors and amino acid properties control the response to Ca binding. Aim 2 involves using the information from Aim 1 to develop hypotheses about interactions that are important for stabilizing different conformations of EF-hand CaBPs, then testing these hypotheses by site-directed mutagenesis experiments. The ultimate goal of these studies is to design, produce and characterize calbindomodulin, a calbindin D/9k mutant whose response to Ca binding has been altered to mimic that of the homologous EF-hand CaBP calmodulin. Measurements of stability, Ca affinity, and hydrophobic affinity will be used to assay mutants, along with a structural screen using NMR. The ultimate goal of functional Ca site design requires the ability to insert a Ca site in a target protein framework, but such methods are not yet available. In Aim 3, we will implement a structure-based approach using the program DEZYMER to design metal sites into green fluorescent protein and photoactive yellow protein. The method will be tested and refined by producing and characterizing mutants that bind Ca, within the context of developing novel Ca biosensors. Our results will contribute to Program-wide efforts to improve the coupling of protein energetics and protein design, and to better understand key common principles for metal site design in proteins, including metal recognition, binding and specificity, additivity of mutations, and long range effects from the protein environment.